Extrasynaptic δ-GABAA receptors are high-affinity muscimol receptors.

Summary

Muscimol, the psychoactive compound from the Amanita muscaria mushroom, binds with high affinity to a specific subtype of GABAA receptors that contain the δ subunit. Deleting the δ subunit in mice eliminates more than 50% of high-affinity muscimol binding sites in the brain, even though δ-containing receptors are relatively scarce. Native δ-GABAA receptors in wild-type mice show binding affinities around 1–2 nM, and incorporating the δ subunit into recombinant receptors produces currents with slow deactivation and extreme sensitivity to muscimol. The findings indicate that δ subunit incorporation dramatically increases muscimol sensitivity, explaining low-dose selectivity for δ-containing receptors in biochemical and behavioral experiments.

Study at a glance

Abstract

Muscimol, the major psychoactive ingredient in the mushroom Amanita muscaria, has been regarded as a universal non-selective GABA-site agonist. Deletion of the GABAA receptor (GABAA R) δ subunit in mice (δKO) leads to a drastic reduction in high-affinity muscimol binding in brain sections and to a lower behavioral sensitivity to muscimol than their wild type counterparts. Here, we use forebrain and cerebellar brain homogenates from WT and δKO mice to show that deletion of the δ subunit leads to a > 50% loss of high-affinity 5 nM [3 H]muscimol-binding sites despite the relatively low abundance of δ-containing GABAA Rs (δ-GABAA R) in the brain. By subtracting residual high-affinity binding in δKO mice and measuring the slow association and dissociation rates we show that native δ-GABAA Rs in WT mice exhibit high-affinity [3 H]muscimol-binding sites (KD ~1.6 nM on α4βδ receptors in the forebrain and ~1 nM on α6βδ receptors in the cerebellum at 22°C). Co-expression of the δ subunit with α6 and β2 or β3 in recombinant (HEK 293) expression leads to the appearance of a slowly dissociating [3 H]muscimol component. In addition, we compared muscimol currents in recombinant α4β3δ and α4β3 receptors and show that δ subunit co-expression leads to highly muscimol-sensitive currents with an estimated EC50 of around 1-2 nM and slow deactivation kinetics. These data indicate that δ subunit incorporation leads to a dramatic increase in GABAA R muscimol sensitivity. We conclude that biochemical and behavioral low-dose muscimol selectivity for δ-subunit-containing receptors is a result of low nanomolar-binding affinity on δ-GABAA Rs.